Running Title: Recent Blastocystis Research
Recent developments in Blastocystis research
C. Graham Clark1, *, Mark van der Giezen2, Mohammed A. Alfellani1,4, C. Rune
Stensvold3
1 – Faculty of Infectious and Tropical Diseases, London School of Hygiene and
Tropical Medicine, London WC1E 7HT, UK
2 - Biosciences, College of Life and Environmental Sciences, University of Exeter,
Exeter EX4 4QD, UK
3 – Statens Serum Institut, DK-2300 Copenhagen S, Denmark
4 - Department of Parasitology, Faculty of Medicine, Sebha University, Sebha,
Libya
*Corresponding Author
Abstract:
Blastocystis is a common parasite of the human large intestine but has an
uncertain role in disease. In this review, we appraise the published evidence
addressing this and its weaknesses. Genetic diversity studies have led to the
identification of numerous subtypes within the genus Blastocystis and, recently,
methods for studying variation within subtypes have been developed, with
implications for our understanding of host specificity. The geographic
distribution of subtypes is summarised and the impact this may have on
investigations into the role of the organism in disease is discussed. Finally, we
describe the organelle and nuclear genome characteristics and look to future
developments in the field.
Key words:
Blastocystis, small subunit ribosomal RNA gene, phylogeny, multilocus sequence
typing, taxonomy, Irritable Bowel Syndrome, genome
1. INTRODUCTION
2. BACKGROUND
3. SUBTYPES
3.1 Current status
3.2 Intra-subtype diversity
4. GEOGRAPHIC VARIATION IN BLASTOCYSTIS PREVALENCE
5. LINKING BLASTOCYSTIS TO DISEASE
5.1 Prevalence and intensity of infection
5.2 Links to Irritable Bowel Syndrome
5.3 Case studies
5.4 The way forward?
6. GENOME STUDIES
6.1 Blastocystis MLO genomes
6.2 Blastocystis nuclear genome
7. FUTURE DEVELOPMENTS
REFERENCES
1. INTRODUCTION
Organisms assigned to the genus Blastocystis are the most common eukaryotes
reported to colonise humans, yet remain an enigma on many levels. Despite
having been described more than 100 years ago (Alexeieff, 1911; Brumpt, 1912),
the question of whether Blastocystis causes disease or is a commensal of the
human gut still has no definitive answer. Our understanding of its taxonomy has
improved but our knowledge of its genetic diversity, host specificity and
geographic distribution remains very incomplete. This review will critically
evaluate the information that has recently become available on the pathogenicity
of this organism, summarise our understanding of its prevalence, diversity and
distribution, and give an overview of the data emerging from genome projects.
2. BACKGROUND
After bouncing between many taxonomic ‘homes’ during most of the 20th
century, Blastocystis finally came to rest among the stramenopiles in 1996
(Silberman et al., 1996), a grouping that did not exist before 1989 (Patterson,
1989). Blastocystis is an atypical stramenopile as this group is named for the
straw-like tubular hairs on the flagella and sometimes the cell body – Blastocystis
has no flagella or tubular hairs. The link was made using phylogenetic analysis of
small subunit ribosomal RNA gene (SSU-rDNA) sequences and has been
confirmed using other gene sequences (Arisue et al., 2002). Within the
stramenopiles, Blastocystis is specifically related to the Proteromonadidae and
Slopalinida (Kostka et al., 2007), which are mostly commensal flagellated or
ciliated organisms found in reptiles and amphibia. They include the genera
Proteromonas, Opalina, Protoopalina, Karotomorpha and Cepedea. The absence of
typical stramenopile morphology in Blastocystis is clearly the result of secondary
loss.
In fact, Blastocystis morphology is not well understood. A large number of
morphological cell types have been described (Zierdt, 1991) but, in the opinion
of the present authors, many are likely to be artifactual and due to oxygen
exposure rather than actually occurring in vivo (Stenzel et al., 1991; Vdovenko,
2000). Nevertheless, it can be stated confidently that Blastocystis is normally a
spherical cell of ca. 5–10 µm in diameter that is multinucleated and contains
multiple mitochondrion-like organelles, Golgi apparatus and other typical
eukaryotic cellular features. Transmission of infection is via a small cyst stage
that is difficult to detect in stool samples (Stenzel and Boreham, 1991) – when
Blastocystis is observed by light microscopy, it is primarily the vegetative form
that is noted. Under the electron microscope, the nucleus has a distinctive
appearance, with a crescent of dark-staining chromatin being seen on one side
(Zierdt, 1991).
The secondary loss of morphology has been responsible for much of the
confusion surrounding species names and host ranges, because all small spheres
look much alike. As a result, species names for Blastocystis were until recently
linked to the host in which they were found – the prime example being
Blastocystis hominis as the name applied to all Blastocystis seen in humans. The
advent of nucleic acid-based analyses in the mid-1990s quickly revealed two
things: 1. The SSU-rDNA from Blastocystis in humans is genetically extremely
diverse; 2. SSU-rDNA from Blastocystis in other hosts can be indistinguishable
from that in humans (Böhm-Gloning et al., 1997; Clark, 1997). This meant that
host origin was not a reliable indicator of organism identity and that some other
means of identifying types of Blastocystis would be necessary.
Over the next 10 years, molecular analyses of Blastocystis became quite popular
and a number of groups in different parts of the world were working
independently to understand the significance of genetic diversity in Blastocystis.
This had an unfortunate consequence, namely that each group came up with its
own nomenclature to denote the Blastocystis molecular types that they detected.
In addition, two distinct methods of analysis were being employed: SSU-rDNA
sequencing and PCR amplification of sequence-tagged sites (STS). The former
has the advantage of generating quantitative data and has the ability to detect
new molecular types as they are uncovered. The latter (Yoshikawa et al., 2004b)
has the advantage of better detection of mixed infections since a separate PCR
reaction is performed to detect each major type, but has the limitation of only
detecting seven known types.
By 2006, the literature had become almost impenetrable for anyone not
intimately involved in Blastocystis typing, and even then it required an ability to
cross-reference between nomenclatures to interpret newly published data.
Adding to the confusion, the identifiable variants were given different names –
genotype, clade, group, subgroup, subtype and ribodeme. As a result, a consensus
was developed and was published in 2007 (Stensvold et al., 2007b). The
consensus relied on multiple types of data being available for some strains (eg.
ribodeme + sequence or STS + sequence) to allow extrapolation of the
nomenclature to other strains that shared the same characteristics. Today, for
the most part (but not always) new publications follow the consensus
nomenclature for ‘subtypes’ of Blastocystis. The proposed replacement of all
names for avian and mammalian species, including Blastocystis hominis, with the
identifier ‘Blastocystis sp.’ followed by the subtype number is also widely
employed. This consensus terminology will be followed throughout this review.
It is perhaps appropriate here to propose a standard approach to subtyping.
While we recognise that DNA sequencing will not be easily available to everyone,
the advantages greatly outweigh those of STS. The method used to develop STS
was sequencing of randomly amplified genome fragments followed by the
development of specific primers. These were then validated by testing them
against a panel of isolates of various subtypes. Now that much more is known
regarding diversity of Blastocystis, we feel that re-validation would be
appropriate. In our hands, the STS primers for subtype 4 only amplify one of the
two clades in this subtype, at least in faecal DNA. As mentioned above, only
known subtypes can be detected and, as more have been named, the specificity
of STS has not been further explored nor the range of detectable subtypes
expanded. STS is certainly of limited use if non-human samples are of interest.
STS typing is also more dependent on interpretation – size and specificity of
bands, for example – than is sequence analysis.
In contrast, SSU-rDNA sequencing is a pan-Blastocystis technique and not limited
to known subtypes. It has been shown that sequencing of the complete gene is
not necessary for accurate subtype classification, as long as a diagnostic region is
used that is known for all subtypes. Several regions of the gene (Fig. 1) have been
used by different authors for this purpose (Parkar et al., 2010; Santín et al., 2011;
Scicluna et al., 2006; Stensvold et al., 2006) but in our hands the specificity of
amplification and ease of sequencing of the ‘barcode’ region (Scicluna et al.,
2006) at the very 5’ end of the gene make it the region of choice for subtyping.
That is not to say it is universally successful. It is likely that all of the primer pairs
used occasionally produce non-specific amplicons, especially when screening
DNA extracted directly from faeces and when the sample is actually negative for
Blastocystis.
The barcode region is by far the best represented in the databases, and the
correct subtype can be identified by BLAST analysis in either GenBank or the
new Blastocystis MLST database (www.pubmlst.org/blastocystis; (Jolley and
Maiden, 2010; Stensvold et al., 2012a). The latter has the added advantages of
automatically assigning allele types to the SSU-rDNA as well as using the
consensus subtype nomenclature (unlike GenBank where the subtype is included
only if one was part of the accession submission and no attempt to impose a
standard nomenclature is made). Because of the occasional problem of non-
specific amplification, it is recommended that samples be screened first for
positivity, where possible, using Real-Time PCR (Stensvold et al., 2012b) before
undertaking subtype identification by sequencing.
3. SUBTYPES
3.1 Current status
The use of numbers has the advantage of allowing new subtypes (STs) to be
assigned to novel sequences as they are discovered. However, this requires a
consensus, which does not as yet exist, on what the requirements are for
designation of new subtypes. We suggest that new subtype assignments be based
on complete or essentially complete SSU-rDNA sequences, not on just a small
piece of the gene like the barcode region, even though this may well be how their
novelty is first identified. In most cases, this should not be a burdensome
requirement, especially if cultures exist, but occasionally primary material might
be limited and inhibitors/non-specific amplicons can interfere with successful
sequencing of some products.
An example of how necessary this approach can be exists in the case of ST13. The
subtype was found in a Quokka (a marsupial) and named by Parkar et al. (2010)
in Australia, who showed it to cluster near ST5 in phylogenetic trees. Their
sequence consisted of the 3’ two-thirds of the SSU-rDNA, over 1,000 bases (Fig.
1). Later, Petrášová et al. (2011) identified an infection in a Colobus monkey in
Tanzania as ST5 based on the sequence of the 5’ one-third of the gene – the
barcode region defined by Scicluna et al. (2006). The Colobus sequence was not
identical to previously-known ST5 sequences but that was the most closely
related subtype in the databases. In actual fact, the Colobus sequence belonged to
ST13. This only became apparent when we compared the Colobus and Quokka
sequences to a complete ST13 sequence we had obtained independently (from a
deer; Alfellani et al., submitted). The overlap between the ‘barcode’ region and
the Parkar sequence is short and shows little variation among related subtypes.
Hence the link between the Petrášová et al. Colobus sequence and the available
ST13 sequence was not obvious.
Nevertheless, agreement on how different a new sequence needs to be before
being considered a novel subtype does not exist at present. In 2007, only nine
subtypes were known and all had been identified in humans. All formed discrete
clades in phylogenetic trees that were supported by maximum posterior
probabilities in Bayesian analyses and very high bootstrap values in maximum
likelihood analyses. The minimum divergence between sequences assigned to
different STs was around 5%. As more hosts are sampled and more sequence
variants are discovered, two things are happening. The amount of known
diversity within existing subtypes is increasing and more potentially new
subtypes are being uncovered that differ by less than 5%. In our opinion, when
the divergence from known subtypes is less than this arbitrary figure, care needs
to be taken before assigning a new subtype number unless substantial sampling
has taken place. The reason for this is exemplified by ST3; in this subtype (and
some others), the most divergent SSU-rDNA sequences differ by almost 3%, yet
the clade itself is strongly supported. If a single new sequence is found that
differs from others in a known clade by just over 3%, one of two things can
happen – 1. Additional sampling may ‘fill in’ the gap between the new branch and
the existing clade, indicating that it is part of the same subtype; or 2. Additional
sampling will identify sequences that are specifically related to the new variant
and do not fall in-between, in which case it can be considered a new subtype.
Initially, new variants will often be represented by a single example and so the
problem remains of what to call them. In our opinion, a new sequence type that
differs by 4% or more can be considered a new ST with confidence. A sequence
that differs by less than 1.5% is most likely to fall within the range of variation of
an established subtype. Those that fall between these cut-off values can be
tentatively assigned new ST numbers subject to confirmation by further
sampling – in a way this resembles the ‘Candidatus’ species names used for
bacteria although we would prefer not to use such a prefix for Blastocystis STs!
Ultimately, like species names, Blastocystis ST designations will be accepted by
those in the field or rejected as synonyms based on further data (see (Boenigk et
al., 2012).
One thing rarely mentioned is that the Blastocystis ST nomenclature refers
exclusively to organisms infecting birds and mammals, as these two host groups
share many of the same subtypes even though some host specificity exists.
Blastocystis is common in reptiles and amphibia, and has been reported from
other hosts, such as insects. The assumption exists that such organisms are
unlikely to overlap with the bird/mammal subtypes because of the different
body temperatures of the hosts. However, this need not always be the case – one
toad sequence reported belongs to ST5 (Yoshikawa et al., 2004a). Nevertheless,
most non-bird/non-mammal Blastocystis have their own species names or at
least do not cluster with bird/mammal subtypes. Should interest lead to greater
sampling of such hosts, perhaps a similar approach to that outlined above will
prove necessary to prevent the nomenclature from becoming unwieldy.
When the consensus subtype nomenclature was developed, nine STs were
recognised. Sampling from a wider range of hosts has led to five additional STs
being published; several additional unpublished STs are known to us. We have
no doubt that many more remain to be uncovered as more hosts and more
individuals within already-sampled hosts are studied. Therefore, to discuss host
specificity at this stage is certainly premature in that we know the picture is
incomplete. However, some general trends are starting to emerge that are worth
commenting on.
Of the nine definite subtypes detected in humans, only four are common – ST1,
ST2, ST3 and ST4. Together, these make up around 90% of all human
Blastocystis in surveys that involve subtyping (Alfellani et al., 2013). These will
be discussed further below. The other subtypes are only sporadically reported in
humans and may well prove to be the result of zoonotic transmission, as they are
mostly much more common in non-human hosts. ST5 is prevalent in livestock,
ST6 and ST7 occur frequently in birds, and ST8 is common in some non-human
primates (NHPs). Of the STs that are rare in humans, only ST9 is yet to be
reported from non-human sources.
Although ST5 is rare in humans, it is found commonly in captive apes, although
not in other NHPs (Stensvold et al., 2009a). Its highest frequency seems to be in
livestock, particularly cattle, pigs, sheep and camels (Stensvold et al., 2009a).
Subtypes 6 and 7 have been detected primarily in ground-dwelling birds, with
only single ST7 samples from a goat and a NHP having been found in non-human
mammalian hosts (Alfellani et al., in press and submitted). Other than in humans,
ST8 appears to be restricted to arboreal NHPs from Asia and South America – it
has not been reported from African NHPs (Alfellani et al., in press). The STs with
numbers above 9 are, as far as is known at present, confined to non-human
hosts. Little experimental work on host-specificity has been performed (Iguchi et
al., 2007) and given that diversity within subtypes may be linked to host range
(see below), the results in this respect must be seen as preliminary.
3.2 Intra-subtype diversity
As discussed above, analysis of SSU-rDNA sequences within certain subtypes has
revealed substantial genetic diversity (Scicluna et al., 2006; Yoshikawa et al.,
2009). Since SSU-rRNA genes are generally highly conserved within species, this
finding suggested that the study of variation within subtypes might lead to
further insights into host range and transmission patterns, as well as potentially
identifying surrogate markers for virulence. In particular, it would help in
determining the relevance of the subtyping system, which could be too crude a
classification tool. Investigations into genetic diversity using non-SSU rRNA
genes have therefore started. A multilocus sequence typing (MLST) system has
been developed for ST3 and ST4 (Stensvold et al., 2012a). This is based on the
sequencing of 5–6 loci in the genome of the mitochondrion-like organelle (MLO;
see below) chosen for the presence of polymorphism. MLST systems for ST1 and
ST2 are currently in development, also based on markers in the MLO. MLST has
been used primarily in bacteria – its utility in eukaryotes is restricted by the fact
that most are diploid or have higher ploidy. The existence of heterozygotes
makes interpretation of the data much more difficult than in haploid organisms.
The organelle genome in Blastocystis is effectively haploid, but this characteristic
was only part of the reason for selecting the MLO as the target for our MLST –
very few nuclear gene sequences were available for Blastocystis at the start of the
project so options were limited!
Application of the MLST system to samples from humans and non-human
primates has already led to some important observations. The phylogenetic tree
obtained from analysis of the SSU-rDNA sequence (the barcode region – Fig. 1;
(Scicluna et al., 2006) is congruent with the one inferred from sequences of loci
in the MLO for both ST3 and ST4 (Stensvold et al., 2012a). Hence, the MLST data
have so far validated the use of the barcode region as a suitable marker for inter-
and intragenetic diversity.
The levels of intragenetic diversity in ST3 and ST4 differ dramatically. MLST
analyses of ST3 isolates showed a high discriminatory index compared to the one
obtained for ST4; in other words most strains can be distinguished by MLST in
ST3 while that is not the case for ST4. ST4 from humans shows a surprising
degree of genetic homogeneity, with most SSU-rDNA and MLO loci sequences
being completely identical between samples (Stensvold et al., 2012a).
Conversely, many SNPs in MLO loci of ST3 are shared by only a few strains or are
unique. Importantly, the differences between ST3 and ST4 are not attributable to
the fact that ST4 samples were almost exclusively from humans, since the vast
majority of the ST3 alleles were detected within the human population. Due to
the homogeneity of ST4, and perhaps also because of the fact that ST4 appears to
be absent or at least very rare in some parts of the world, we speculate that ST4
entered the human population relatively recently compared to ST3. ST4 is
common in Europe, but is rarely reported from Asian, Middle Eastern and South
American populations; however, in many regions comparatively little sampling
has been undertaken.
ST3 is the most common subtype in humans worldwide, and its occurrence is a
frequent finding in analyses of subtype distribution, irrespective of the
geographic origin of the population (Forsell et al., 2012; Malheiros et al., 2011;
Meloni et al., 2011; Nagel et al., 2011; Souppart et al., 2010; Souppart et al., 2009;
Stensvold et al., 2009a; Stensvold et al., 2009b; Stensvold et al., 2011b). The high
discriminatory index of the ST3 MLST system makes it useful for surveillance of
ST3 strains (re-infection or recrudescence; longevity of colonisation; patterns of
transmission), whereas that of ST4 would not be suitable for these purposes.
Nevertheless, two clades can be detected in ST4 SSU-rDNA and MLST analyses,
one of which consists mostly of non-human samples. Little is known about the
host range of ST4 but, in addition to humans, it has been found in rodents and
occasionally in non-human primates (lemur (Santín et al., 2011; Stensvold et al.,
2009) and woolly monkey (Alfellani et al., in press)).
Interestingly, MLST analysis of ST3 isolates from humans and non-human
primates revealed that NHP ST3s are significantly more diverse than human
ST3s, most of which fell into one clade. Human ST3 samples are found only rarely
in the ‘NHP clades’ and are likely a result of zoonotic transmission, illustrated by
the fact that one of the few such human isolates was from a NHP keeper. In
contrast, NHP samples were also detected in the clade containing the vast
majority of the human sequences. Together, these results suggest that ST3 has
largely co-evolved with humans, but that either this co-evolution has been going
on for a long time or ‘human clade’ ST3 has entered the human population
repeatedly from another source. This is in contrast to ST4, where the same
restriction of human samples to one clade exists but little sequence diversity is
detected, a finding that implies a single and relatively recent origin in humans
followed by clonal expansion. ST3 has also been reported from a number of non-
primate hosts and MLST analysis of such ST3s is needed to increase our
understanding of the currently observed cryptic host specificity and thus the
transmission and epidemiology of Blastocystis. Both clades of ST4 have been
detected in rodents but relatively little sampling has been reported, so it is not
yet clear whether these hosts are a significant reservoir for human colonisation.
4. GEOGRAPHIC VARIATION IN BLASTOCYSTIS PREVALENCE
The prevalence of Blastocystis is reported in many parasite surveys performed
across the world. Published infection rates fall anywhere between 0.5% and
62%. A serious problem with such data is the often highly selected nature of the
population studied. Only rarely are the surveys large enough or the population
examined diverse enough to really conclude that the infection rate reported is
representative of the country/cohort as a whole, yet that is often how the data
are interpreted. A good illustration of this is where more than one survey has
been performed in the same country. For example, two studies carried out in
Malaysia found prevalences of 15% (Suresh et al., 2001) and 52% (Noor Azian et
al., 2007), while two in Turkey reported 2% (Köksal et al., 2010) and 14% (Ostan
et al., 2007). In Turkey, the studies were undertaken in different cities. One
surveyed school children (Ostan et al., 2007), many of whom lived in a shanty
town, while the other surveyed adults. The diagnostic techniques used, however,
were basically the same. In Malaysia, the age range of the populations was
similar but one population lived in apartments in the capital (Suresh et al., 2001)
while the other (Noor Azian et al., 2007) was in an aboriginal village settlement.
In one of these Malaysian studies, a variety of techniques - including culture -
were used for diagnosis of infection, while the other involved microscopy only. In
both countries, the populations were demographically quite different in several
ways and whether either could be viewed as truly representative of the country
as a whole is debatable. The diagnostic technique used is potentially another
significant variable that can influence the reported prevalence (fresh stool vs.
fixed and concentrated; bright field vs. stained) as microscopy is generally
thought to be less sensitive than culture, although the skill of the microscopist is
also a significant factor. In the future, it seems likely that diagnostic PCR will
become the tool of choice where it can be afforded, which will make comparison
of results with studies using only microscopy even more difficult.
5. LINKING BLASTOCYSTIS TO DISEASE
5.1 Prevalence and intensity of infection
Like the simple population prevalence surveys, many comparisons of Blastocystis
prevalence in symptomatic and asymptomatic individuals have been published.
Approximately equal numbers of papers report significantly higher prevalences
of Blastocystis in symptomatic individuals and no significant difference at all.
Here, some of the same variables are notable confounding factors – how can
studies be compared in which researchers have variously used fixed material or
fresh, direct or concentrated samples, iodine, Trichrome, or haematoxylin as the
stain for microscopy? Furthermore, shedding of Blastocystis may be cyclical
(Vennila et al., 1999), yet it is unusual for more than one stool sample to be
examined. However, when the aim is to explore links to disease, the main
complication is again the selection of the populations – in this case, the
definitions of symptomatic and asymptomatic and comparability of the
symptomatic group and the asymptomatic controls. Most, if not all,
investigations have been cross-sectional, meaning that carriers will have
harboured Blastocystis for different periods of time, which may affect whether
they are experiencing symptoms – if acquired immunity to Blastocystis plays a
role in symptom resolution.
Since Blastocystis is a faecal-orally transmitted parasite, carriers will also have
been exposed to other intestinal organisms, some of which may be pathogens, at
the time of Blastocystis colonisation. Most association studies do not exclude all
other possible origins of the symptoms. Likewise, most control populations are
not case-matched and there is some suggestion that ‘asymptomatic’ individuals
who volunteer for such studies do not represent a random selection but may
have a history of intestinal problems (Stensvold et al., 2011a; Stensvold et al.,
2009b). However, one of the most peculiar variables used is the fact that some
studies define colonisation with Blastocystis as having more than 5 organisms
per microscope field. The rationale for this is unexplained –why would having 4
vs. 6 organisms per field be a significant difference? The recent descriptions of
Real-Time PCR diagnostic tools for Blastocystis (Poirier et al., 2011; Stensvold et
al., 2012b) will make detection of the organisms easier as well as allowing
exploration of any role for infection intensity in symptomatology.
One might assume that animal models are an obvious way of potentially
establishing a link between Blastocystis and pathology. However, the studies
performed to date are, in our opinion, inconclusive. For example, experimental
infections of laboratory mice (Elwakil and Hewedi, 2010) resulted in tissue
invasion – something never reported in humans. Another study showed
increased oxidative stress in Blastocystis -infected rats (Chandramathi et al.,
2010), again something not linked to human colonisation. Studies that provided
evidence for induction of cytokines, contact-mediated apoptosis, and barrier
disruption all used axenic Blastocystis and in vitro mammalian cell cultures, with
no evidence provided that these effects occur in vivo. One other issue is the use
of appropriate controls – for example, experimental infection of animals with
Blastocystis from cultures growing in the presence of bacteria need to have the
appropriate controls – namely, exposure to the accompanying bacterial flora
alone – before it can be concluded that Blastocystis is responsible for any effects
seen (Hussein et al., 2008). It has to be said that, to date, animal models are not
showing much promise in resolving the question of the pathogenic potential of
Blastocystis.
5.2 Links to Irritable Bowel Syndrome
One of the popular associations made in the literature is between Blastocystis
and Irritable Bowel Syndrome (IBS; (Poirier et al., 2012). There are two reasons
for this. The most telling is that people diagnosed with IBS appear in several
studies to have a much higher infection rate with Blastocystis – often twice as
high or more (Giacometti et al., 1999; Jimenez-Gonzalez et al., 2012; Yakoob et
al., 2010; Yakoob et al., 2004). The second is that many of the symptoms ascribed
to Blastocystis infection are very similar to those defining some types of IBS
(diarrhoea, vomiting, abdominal cramps and bloating), suggesting either that
Blastocystis colonisation may be a differential diagnosis or that Blastocystis is the
causative agent in some cases of IBS. Alternatively, it could mean that Blastocystis
colonises the IBS gut more efficiently than a healthy gut. As mentioned above, the
presence of Blastocystis means exposure to faecal organisms and so superficially
at least the data support a link between faecal exposure and IBS rather than a
specific link to Blastocystis.
A diagnosis of IBS should include the exclusion of other potential causes of the
symptoms, but it is not always possible to tell from publications whether this has
been done and what pathogens have been excluded – it is unlikely that every
possible intestinal disease agent has been tested for. IBS is also not a disease
with a unique and specific diagnosis; it is a syndrome having several different
forms. Diagnosis is currently based on the Rome III criteria: “Recurrent
abdominal pain or discomfort at least 3 days per month in the last 3 months
associated with 2 or more of the following
Improvement with defecation
Onset associated with a change in frequency of stool
Onset associated with a change in form (appearance) of stool”
The “change in frequency” covers diarrhoea (IBS-D) and constipation (IBS-C) or
alternation of the two (IBS-M), yet in several Blastocystis studies these are not
differentiated.
One illustration of the problem can be found in the study by Giacometti et al. in
Italy (1999). The authors compared Blastocystis prevalence in IBS patients
(15/81) to that in patients with other gastrointestinal complaints (23/307) and
found it to be significantly different (p=0.006). However, when the IBS patients
were followed up 6 months later, 53/72 returning patients no longer met the
criteria for a diagnosis of IBS. Since IBS is considered a chronic disease, this calls
into question the original diagnosis as such a high rate of ‘cure’ would not be
expected.
In a study in Pakistan, Hussain et al. (1997) found that levels of antibody against
Blastocystis were higher in patients with IBS than in controls but, surprisingly,
levels in IBS patients were the same whether the parasite was detected
concurrently or not. In Mexico and Thailand, three studies failed to show a
higher prevalence of Blastocystis in IBS patients (Ramirez-Miranda et al., 2010;
Surangsrirat et al., 2010; Tungtrongchitr et al., 2004).
One of the factors that make the interpretation of all these studies of
Blastocystis/IBS prevalence and association difficult is the existence of nine
subtypes in humans that, as judged by molecular criteria, could be considered
distinct species. More than 20 countries have been surveyed for the range of
subtypes present and this number is gradually increasing. Differences in the
subtypes present in a country could be responsible for the differing conclusions
from research investigating links between Blastocystis and disease. However,
only a small number of studies to date have compared distribution of subtypes in
symptomatic and asymptomatic individuals.
From the geographic subtype surveys (Table 1), several interesting observations
have emerged. The first is a technical one. Surveys that use the STS method of
subtyping detect ST6 and ST7 at a much higher frequency on average than those
using SSU-rDNA sequencing. It is difficult to interpret this, however, as the STS
method is most widely used in continental Asia and there are no comparable
sequencing studies in the same countries that can be compared to STS. The
exception to this disjunction is Egypt, where two STS studies found many ST6
and ST7 (48/144) but sequencing did not (0/20). Nevertheless, it is possible that
ST6 and ST7 are simply more common in humans in Asia.
The second observation has to do with the distribution of ST4. This subtype is
very common in Europe (often the second most common ST found after ST3), but
apparently absent in Egypt, Libya, Iran, Nepal, the Philippines, Thailand, Malaysia
and Brazil, and rare in many other countries (see summary in Alfellani et al.,
2013). Again, there may be some link to the method used (STS in Asia) but not in
all countries as sequencing was used in the studies from Libya and Brazil, for
example. One possible explanation would be an inability of the STS method to
amplify certain clades within subtypes (as discussed earlier). However, if this is
the case, more unidentified Blastocystis isolates would be expected in STS
analysis, and these are not commonly reported. Thus, it appears that ST4 has a
very uneven distribution across the world, with the focus being in Europe –
perhaps that is where this subtype entered into the human population in the
recent past – while STs 6 and 7 are rare in humans outside of Asia. There is no
immediately obvious reason why humans in Europe might be more exposed to
rodents and those in Asia to birds, so zoonotic transmission seems an unlikely
explanation. Perhaps some dietary, cultural or other demographic variables are
responsible – at present, it is not useful to speculate.
Three studies have investigated the possibility of a link between Blastocystis
subtypes and IBS. However, the results are very inconsistent. In Pakistan
(Yakoob et al., 2010) and Egypt (Fouad et al., 2011) significant differences in
subtype distribution were found, with ST1 being much more common in IBS
patients than controls. A study in the UK found ST4 to be more common and ST1
less so in patients from IBS clinics than in other diagnostic laboratory samples,
but not reaching statistical significance (Alfellani et al., 2013). Again, there was a
difference in methodology (STS vs. sequencing) as well as many other variables,
not the least of which is geography and the presence of ST4, so the potential
relationship needs further investigation. What can be concluded is that no single
subtype is found in IBS patients. Perhaps this is not surprising, given the
variability in the combination of symptoms that can lead to a diagnosis of IBS.
5.3 Case studies
The other source of information on the link between subtype and symptoms has
been individual case studies. In such reports, an individual with gastrointestinal
symptoms, Blastocystis infection, and no other identifiable cause is investigated
and treated. Often the report correlates clearance of the parasite with resolution
of symptoms and, in the recent past, the subtype of the organism present has
often been determined. There are two problems with such reports. The first is
that the drugs used to ‘eliminate’ Blastocystis are many and varied but have no
known specificity for the parasite. It seems equally likely that the treatment
perturbs the intestinal flora, indirectly making it a poor or unsuitable habitat for
Blastocystis. The second problem is that all common subtypes and certain others
have been linked with symptoms, which seems an unlikely situation (Dogruman-
Al et al., 2008; Domínguez-Márquez et al., 2009; Jones et al., 2009; Stensvold et
al., 2008; Stensvold et al., 2011a; Vassalos et al., 2010; Vogelberg et al., 2010).
Treatment of Blastocystis colonisation is contentious and no widely accepted
drug regimen exists (see (Stensvold et al., 2010). It is certainly not surprising
that subtypes differ in their susceptibility to drugs, given the large genetic
differences that exist between them (Mirza et al., 2011), and in vitro drug
resistance has been generated successfully in the laboratory (Dunn et al., 2012).
5.4 The way forward?
To summarise current data on the relationship between Blastocystis and disease
is not easy, since we feel that the definitive investigations are yet to be carried
out. Because of the valid argument that if disease is associated with one subtype,
the signal may be masked when the overall prevalence in a population is
considered, we believe that: it is necessary for subtyping to be performed;
sequencing should be used if possible in preference to the indirect STS method;
and DNA extracted directly from faeces should be used in preference to cultures,
in case cultivation selects in favour of certain subtypes. It is possible that
Blastocystis is a pathogen but that this is unlinked to the subtype of the organism.
Nevertheless, subtyping should be done if only to rule this out as a variable. It is
also important that studies be carried out in more than one country, given the
apparent geographic differences in subtype distribution. For example, ST4 is
prevalent in Europe and has been linked to symptoms in more than one
investigation. If ST4 is the only subtype linked to disease, a study in a country
where the subtype has not been reported or is rare will not reveal an association
of Blastocystis with disease even if one exists.
The appropriate study population is difficult to define. The presence of 4
common subtypes (in Europe at least) means that more individuals are needed
in order to have the same power to detect an association. Most crucial of all are
the faecal samples themselves. An advantage of using IBS patients is that they
have usually had an extensive microbiological workup to eliminate other
pathogens as a cause of their symptoms, which is a deficiency in many other
studies. However, it seems unlikely that if Blastocystis causes IBS, it is
responsible for both IBS-D and IBS-C. The question remains of how to obtain a
suitable control group for IBS patients.
In many cultures, people are very happy to provide blood samples but reluctant
to donate faecal specimens unless they are ill, which has impaired the ability of
controlled studies to be undertaken in many countries. Nevertheless, research
has been successfully undertaken where a large number of individuals with
gastrointestinal symptoms and a similarly large asymptomatic population of
individuals have been sampled for investigation of intestinal pathogens (eg.
(Tam et al., 2012). Unfortunately, Blastocystis has not been included in those
studies.
An alternative approach is to identify a smaller number of individuals infected
with Blastocystis but no other potential pathogen, and then match these as
closely as possible with asymptomatic controls. Recruitment of individuals from
web panels (for instance, the ‘YouGov’ panel in Denmark) has previously been
used successfully to obtain data on the prevalence of gastrointestinal symptoms
in the background population (Reimer and Bytzer, 2009). Again, the presence of
4 common subtypes means that the sample numbers needed might make this
approach difficult. It is also possible that symptoms resulting from Blastocystis
infection are acute but resolve, and we have no knowledge of how long
Blastocystis colonisation may persist for afterwards. Until appropriate studies
are performed and a clear answer obtained, the question of whether Blastocystis
is a pathogen will continue to be contentious.
Whatever the ultimate outcome, it must be emphasised that infection with
Blastocystis is a surrogate marker for exposure to faecal contamination, and in an
individual with symptoms and Blastocystis colonisation, an infectious agent
(whether it is Blastocystis or not) seems the most likely cause.
6. GENOME STUDIES
Other than investigations into Blastocystis diversity and the role of the organism
in disease, the most important recent advances have been those involving
genome studies. These consist of projects that have given rise to the sequences of
one nuclear genome and several mitochondrial genomes. The latter represent a
number of different subtypes and, as mentioned earlier, these sequences have
been the basis for development of MLST schemes for the most common subtypes.
6.1 Blastocystis MLO genomes
The presence of mitochondrion-like organelles in Blastocystis was for many
years an enigma as it was unclear why a strictly anaerobic eukaryote needs an
organelle traditionally associated with aerobic metabolism. Nevertheless, it was
shown quite early on using DNA stains that these organelles contain DNA and
this attracted the attention of those interested in organelle evolution. The
presence of mitochondrion-derived organelles had been the subject of
investigation for some years but it became clear that the mitosomes of
Entamoeba, microsporidia and Giardia and the hydrogenosome of Trichomonas
are actually quite different end products of reductive evolution from the
mitochondrial endosymbiont – and none have retained any trace of a genome in
the organelle (van der Giezen, 2009). The possibility that the Blastocystis MLO
represents an intermediate step in the ‘degeneration’ of the mitochondrion is
intriguing.
Three MLO genomes were published almost simultaneously, representing by
chance three different subtypes, one from each of the three main clades of
human Blastocystis: ST1, ST4 (Pérez-Brocal and Clark, 2008) and ST7
(Wawrzyniak et al., 2008). All three encode the same 27 proteins and 18 RNAs,
and the gene order is identical. The 27.7–29.2kb genomes were found to contain
several nad genes, encoding proteins of mitochondrial Complex I, and ribosomal
protein genes; none of the genes encoding cytochromes and ATPase subunits
found in other stramenopile genomes are present. A reduced set of tRNA genes
identified in the Blastocystis MLO genome implies that tRNAs for some codons
must be nuclear encoded and imported from the cytoplasm.
6.2 Blastocystis nuclear genome
Of all stramenopile nuclear genomes sequenced to date, the one from Blastocystis
strain B (ST7) is the smallest. It is just under 19 Mb in size and contains about
6,000 genes, which is just over half the number in the stramenopile genome with
the next lowest number of genes (see Table 2). It is also relatively intron rich, but
its introns are by far the smallest found among stramenopiles as the median size
is only 32 bp (Denoeud et al., 2011). Although it is useful to make comparisons
with other stramenopile genomes, those genomes available are of relatively
distant species and none are for human pathogens. Although the Phytopthora
and Pythium genomes can provide some useful reference material because of the
pathogenic nature of these organisms (in plants), it should not be forgotten that
Blastocystis is the only anaerobe among the sequenced stramenopiles, and hence
the special features of its genome could have been driven by environmental
factors as well as being reflective of its evolutionary distance from the other
sequenced stramenopiles.
Nonetheless, it might be useful to look at the presence of potential effector
proteins encoded in the Blastocystis genome and other proteins that might play a
role in pathogenesis. Such analyses might provide useful avenues for exploring
the potential pathogenicity of this organism. Many eukaryotic pathogens use
effector proteins to remodel their host’s cells/tissues into more suitable niches
for proliferation. If Blastocystis is truly a pathogen, then one might expect it to
use strategies similar to those of other eukaryotic pathogens, perhaps in
particular those used by Phytophthora species. Effectors are molecules that
either facilitate infection (virulence factors or toxins) or that trigger host defence
(avirulence factors or elicitors) (Kamoun, 2006). In order to be able to affect the
host, these effectors need to be secreted by the pathogen, and analysis of the
Blastocystis genome using SignalP has suggested that 307 proteins contain
secretion signals (Denoeud et al., 2011). Whether these potentially secreted
proteins contain any additional host cell targeting signals, such as the
Plasmodium RxLxE/D/Q motif or the Phytophthora infestans RxLR motif (Haldar
et al., 2006) still needs to be investigated. Among the proteins making up the
putative Blastocystis secretome are hydrolases, proteases and protease
inhibitors. The latter are generally involved in protecting parasite proteins from
degradation. The Blastocystis genome encodes a cystatin A homologue, a type–1
proteinase inhibitor and an endopeptidase inhibitor-like protein (Denoeud et al.,
2011). However, only one of these seems to encode a putative secretion signal,
using SignalP, and none contain the Kazal-like domains that are often found in
secreted protease inhibitors of eukaryotic parasites (Haldar et al., 2006). The
genome is predicted to encode several hydrolases that might be involved in
attacking host tissue (Denoeud et al., 2011), although tissue invasion by
Blastocystis has never been observed in humans. The most likely possible
effectors found in the Blastocystis genome are cysteine proteases, considering
that these genes are also present in large numbers in the pathogenic protist
Entamoeba histolytica (Bruchhaus et al., 2003). Nonetheless, these predictions all
need further elucidation in the laboratory in order to determine whether they
have any role in pathogenesis and disease and to prove that they are secreted.
Two secreted proteases have recently been characterised (Wawrzyniak et al.,
2012). It is anticipated that more subtypes will have their genomes sequenced in
the near future. This will help to confirm that the oddities of the ST7 genome
apply to all Blastocystis and are not subtype-specific, and so are relevant to the
common human-infective subtypes.
In common with other protistan genomes (Carlton et al., 2007; Loftus et al.,
2005), Blastocystis seems to contain a number of genes that may have been
acquired by lateral gene transfers (LGT). Two possible red algal genes might hint
at a lost chromalveolate plastid while others might be involved in some aspects
of anaerobic fermentation (Denoeud et al., 2011). The anaerobic nature of
Blastocystis combined with the presence of MLOs with cristae that are capable of
taking up active dyes such as Rhodamine 123 (Nasirudeen and Tan, 2004) has
sparked an interest in the nature of these organelles (Denoeud et al., 2011;
Lantsman et al., 2008; Stechmann et al., 2008). Unlike classic mitochondria, the
Blastocystis organelles contain the enzymatic capability to convert pyruvate into
CO2 and H2 using enzymes normally encountered in hydrogenosomes (van der
Giezen, 2009). Although hydrogen production has not been detected (Lantsman
et al., 2008), the enzyme hydrogenase does localise to the organelle (Stechmann
et al., 2008). Furthermore, the organelle contains the unusual acetate: succinate–
CoA transferase (ASCT) shuttle, which allows for the production of ATP via
substrate-level phosphorylation. The absence of cytochromes had been reported
early on (Zierdt, 1986) so the lack of mitochondrial Complex III and IV
components in the genome came as no surprise. Many genes encoding proteins
that make up Complex I, possibly involved in proton pumping, have been
detected, as has a complete Complex II (Denoeud et al., 2011; Stechmann et al.,
2008). As no further downstream electron transport chain components have
been found, the question whether Complex II functions as a succinate
dehydrogenase, as in classical mitochondria, or as a fumarate reductase, is still
open. Because of the anaerobic nature of Blastocystis, a fumarate reductase using
rhodoquinone seems a plausible possibility. Currently, the terminal electron
acceptor (Denoeud et al., 2011) seems to be the alternative oxidase (Standley
and van der Giezen, 2012) but the choice of molecular oxygen as a substrate
seems odd for an intestinal organism.
Overall, the complete genome of Blastocystis (Denoeud et al., 2011) has
confirmed many previous studies (Lantsman et al., 2008; Stechmann et al., 2008;
Zierdt, 1986; Zierdt et al., 1988) with respect to the biochemical nature of the
MLO but several questions still remain. Perhaps the most significant one relates
to the anaerobic status of this organism (Zierdt, 1986), as its genome suggests
that it most likely is not a strict anaerobe after all.
7. FUTURE DEVELOPMENTS
It is probable that new genomes will become available in the immediate future,
both from MLOs and from nuclei of different subtypes, and hopefully also from
non-bird/non-mammalian Blastocystis as well, so that features common to
Blastocystis can be distinguished from those that may be lineage-specific
adaptations. We fully expect that surveys of Blastocystis subtypes from
previously unsampled regions of the world will be forthcoming. The current
impression of subtype distribution being geographically disjunct might be
affirmed by such studies, or they may result in subtype prevalence numbers
being seen as part of a continuum from common to absent. Further sampling of
an increasing range of non-human hosts will also help confirm or refute the
current impression of partial host-specificity of certain subtypes and genotypes.
The situation regarding the role of Blastocystis in disease is more difficult to
predict as the necessary investigations will be expensive and time-consuming to
conduct in a way that will give unambiguous answers. They will also have to be
carried out in different parts of the world, given the observed geographic
variation in subtype prevalence. Nevertheless we feel that these are the most
important types of study to perform, because until the uncertainty surrounding
the role of Blastocystis in disease is settled, it seems likely that it will continue to
be dismissed by many clinicians as an organism of no importance. In the
meantime, screening by Real-Time PCR and barcode-sequencing of Blastocystis
in human cohorts with varying symptoms and different geographic origins will
continue to provide useful prevalence, subtype and parasite load data.
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Country of samples
Technique* No. of
samples
Subtype distribution
Reference ST1 ST2 ST3 ST4 ST5 ST6 ST7 ST8 ST9
Mixed ST
Unknown ST
Germany RFLP 166 35 1 110 12 — — — — — 8 — Böhm-Gloning et al., 1997
UK RFLP 29 2 1 22 4 — — — — — — — Clark, 1997
Japan STS 32 1 — 30 — — 1 — — — — — Yoshikawa et al., 2000
Japan RFLP 64 11 13 30 7 — — — — — — 3 Kaneda et al., 2001
Thailand RFLP 153 138 — 7 — — — 2 — — 6 Thathaisong et al., 2003
Japan STS 50 4 — 26 2 — 11 5 — 2 — — Yoshikawa et al., 2004b
Bangladesh STS 26 2 — 24 — — — — — — — — Yoshikawa et al., 2004b
Pakistan STS 10 2 — 7 — — 1 — — — — — Yoshikawa et al, 2004b
Germany STS 12 3 2 5 2 — — — — — — — Yoshikawa et al., 2004b
Thailand STS 4 1 — 1 — — 1 — — — 1 — Yoshikawa et al., 2004b
Philippines RFLP 12 10 — — — — — — — — — 2 Rivera and Tan, 2005
Denmark Sequencing 29 1 6 15 7 — — — — — — — Stensvold et al., 2006
UK Sequencing 49 2 8 20 16 — — 1 1 — 1 — Scicluna et al., 2006
China STS 35 13 — 14 — — — 2 — — 5 1 Yan et al., 2006
Denmark Sequencing 28 5 9 13 1 — — — — — — — Stensvold et al., 2007a
China STS 192 47 9 116 1 — 1 — — — 10 8 Li et al., 2007
Egypt STS 44 8 — 24 — — 8 4 — — — — Hussein et al., 2008
Greece SSCP 45 9 6 27 1 — 1 1 — — — — Menounos et al., 2008
Malaysia STS 20 9 1 10 — — — — — — — — Tan et al., 2008
Ireland Sequencing 14 1 6 4 3 — — — — — — — Scanlan and Marchesi, 2008
Iran RFLP 45 20 4 16 — — — — — — — 5 Motazedian et al., 2008
Turkey Sequencing 87 8 12 66 1 — — — — — — — Özyurt et al., 2008
Singapore RFLP 9 2 — 7 — — — — — — — — Wong et al., 2008
Turkey STS 92 17 20 51 — — — — — — 4 — Dogruman-Al et al., 2008
Spain RFLP 51 1 2 — 48 — — — — — — — Domínguez-Márquez et al., 2009
France Sequencing 40 8 4 20 4 — — 1 — — 3 — Souppart et al., 2009
Nepal STS 20 4 4 12 — — — — — — — — Yoshikawa et al., 2009
Malaysia STS 40 5 — 20 — — 11 2 — — — 2 Tan et al., 2009
Turkey STS 32 20 3 9 — — — — — — — — Eroglu et al., 2009
Denmark Sequencing 99 20 15 39 16 — 1 — 1 — 7 — Rene et al., 2009
Denmark Sequencing 116 21 22 21 20 — — 5 — 1 26 — Stensvold et al., 2009b
Turkey STS 19 0 8 10 — — — — — — 1 — Dogruman-Al et al., 2009a
Turkey STS 66 10 9 38 — — — — — — 9 — Dogruman-Al et al., 2009b
Egypt Sequencing 20 3 4 12 — — — — — — 1 — Souppart et al., 2010
Pakistan STS 179 87 10 49 8 7 6 10 — — — 2 Yakoob et al., 2010
Turkey STS 25 9 6 10 — — — — — — — — Eroglu and Koltas, 2010
France Sequencing 27 1 1 4 17 — 1 3 — — — — Poirier et al., 2011
Colombia Sequencing 12 4 3 4 - — — — — — 1 — Santín et al., 2011
Denmark Sequencing 25 1 4 1 19 — — — — — — — Stensvold et al., 2011a
Denmark Sequencing 22 9 11 — — — — — — — 2 — Stensvold et al., 2011b
Italy Sequencing 30 2 5 13 6 — — — — — 4 — Meloni et al., 2011
Brazil Sequencing 66 27 21 11 7 Malheiros et al., 2011
Egypt STS 100 15 — 39 — — 23 13 — — 10 — Fouad et al., 2011
Sweden Sequencing 63 10 9 30 13 — — 1 — — — Forsell et al., 2012
Total 2299 608 239 987 208 7 66 50 2 3 106 23
Table 1. Subtype geographic distributions. Reports using different techniques and older terminologies have been translated into the
consensus terminology of subtypes (Stensvold et al., 2007a). *Technique: RFLP = Restriction Fragment Length Polymorphism; SSCP =
Single Strand Conformation Polymorphism; Sequencing = partial or complete SSU-rRNA gene.
Species Size
(Mb) Chromosomes Genes Average
gene length
GC content
Introns Average intron length
Introns/ gene
Reference
Blastocystis sp. 18.8 15 6020 1299 - 18 560 32 bp 3.1 (Denoeud et al., 2011)
Ectocarpus siliculosis
195.8 - 16 256 6859 54% 113 619 704 bp 6.98 (Cock et al., 2010)
Thalassiosira pseudonana
34.3 24 11 242 992 47% 15 739 - 1.4 (Armbrust et al., 2004)
Phytophthora infestans
240 8-10 17 797 1523 51% - 125 bp - (Haas et al., 2009)
Phytophthora sojae
95 - 19 027 1613 54% - 124 bp - (Tyler et al., 2006)
Phytophthora ramorum
65 - 15 743 1625 54% - 123 bp - (Tyler et al., 2006)
Phaeodactylum tricornutum
27.4 33 10 402 - - 8169 - 0.79 (Bowler et al., 2008)
Pythium ultimum
42.8 - 15 290 - 52% 24 464 115 bp 1.6 (Lévesque et al., 2010)
Table 2. Comparison of several features of stramenopile genomes. Several more stramenopile genome projects are currently ongoing but not all information for inclusion in this table is readily available. ‘-‘ = data not available.
Figure legend:
Figure 1. Schematic representation of the Blastocystis SSU-rRNA gene. Examples
of the regions of the gene used for subtype identification by various authors are
indicated (Parkar et al., 2010; Santín et al., 2011; Scicluna et al., 2006; Stensvold
et al., 2006).
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